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目的:建立大鼠耳蜗大、小上皮嵴细胞永生细胞系。方法:取P1大鼠耳蜗,分离出耳蜗上皮薄片组织,并与含SV40-LT抗原的逆转录病毒共培养,从而实现细胞的永生化。利用单克隆法纯化细胞系,并通过形态学、免疫组织化学等方法对细胞系进行鉴定。结果:大鼠耳蜗大、小上皮嵴细胞永生细胞系基本保持了细胞原代的表型特征:呈现上皮细胞样的多角形外观;不同世代的细胞均表达Islet1及SV40-LT抗原。该系目前已培养3个月,超过20代,传代时间为3~4d,传代比例为1∶5。结论:本实验成功建立了大鼠耳蜗大、小上皮嵴细胞的永生细胞系,这为大、小上皮嵴细胞生物学特性及相关功能,特别是毛细胞再生及前体细胞移植治疗感音神经性聋方面的研究奠定了坚实的实验基础。
Objective: To establish immortal cell line of rat cochlear large and small epithelial crest cells. Methods: The cochlea of P1 rat was isolated and cochlear epithelial tissue was isolated and co-cultured with retrovirus containing SV40-LT antigen to achieve immortalization of cells. Cell lines were purified by the monoclonal method and cell lines were identified by morphological, immunohistochemical and other methods. RESULTS: The immortalized cell lines of cochlear large and small epithelial cells of the rat basically maintained the phenotypic characteristics of the cells. The epithelial cells showed polygonal appearance. The cells of different generations expressed Islet1 and SV40-LT antigens. The Department has been cultured for 3 months, more than 20 generations, passage time is 3 ~ 4d, passage ratio of 1: 5. CONCLUSIONS: The immortal cell line of large and small epithelial crest cells of rat cochlea was successfully established in this experiment. This is the biological characteristics and related functions of large and small epithelial crest cells, especially hair cell regeneration and precursor cell transplantation for the treatment of sensorineural nerve Research on deaf has laid a solid experimental foundation.